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New method for detecting SARS antibody in serum sample and a product thereof

A technology for detecting antibodies and samples, applied in the field of new detection of SARS antibodies and products in serum samples, can solve the problems of lack of models and evaluation, and achieve the effect of good consistency

Inactive Publication Date: 2009-09-16
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, whether the suspension chip can detect SARS antibodies in human serum still lacks models and evaluations

Method used

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  • New method for detecting SARS antibody in serum sample and a product thereof
  • New method for detecting SARS antibody in serum sample and a product thereof
  • New method for detecting SARS antibody in serum sample and a product thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0044] 2. Preparation of samples to be tested

[0045] 1. Preparation of target analyte samples

[0046] The target analyte is rabbit anti-SARS IgG, and the interfering sample or the sample used as method-specific test is other antibodies or other proteins other than the target detection object, including rabbit anti-tuberculosis serum, rabbit anti-influenza NP IgG, rabbit anti-avian influenza H5N1 serum, Rabbit anti-plague F1 IgG, BSA, casein, tryptone, etc. All the above-mentioned samples to be analyzed were dissolved in the sample diluent and stored at 4°C. The stock solution concentration of rabbit anti-rabbit anti-SARS N IgG is 0.036mg / mL. In the comparison experiment, the same sample was used for the detection of ELISA and suspension chip.

[0047] Dilute the rabbit anti-SARS IgG sample diluent to be analyzed into samples of different concentrations in a 4-fold ratio to draw a standard curve for sample detection dose-response. Among them, the concentrations of several...

Embodiment 1

[0050] Embodiment 1, the preparation of the protein suspension chip that detects SARS antibody

[0051] 1. Capturing antigen-coated encoded microspheres

[0052] The No. 044 coded microspheres used in the present invention were purchased from BIO-RAD Company of the United States. The coded microspheres are used to label the SARS-CoV N protein antigen that can capture SARS antibodies, that is, the SARS-CoV N protein is used to coat the microspheres.

[0053] A. Activation of encoded microspheres

[0054] Take 100μL (1.25×10 6 pcs) encoded microspheres into a 1.5mL centrifuge tube, centrifuge at 14000×g, carefully aspirate and discard the supernatant. Add 100 μL of microsphere washing buffer to suspend, shake and sonicate, centrifuge at 14000×g, carefully aspirate and discard the supernatant. Add 100 μL of microsphere activation buffer, then add 10 μL of freshly prepared EDC (50 mg / mL), then add 10 μL of freshly prepared 50 mg / mL Sulfo-NHS, shake at high speed for 30 seconds,...

Embodiment 2

[0064] Embodiment 2, optimization of suspension chip preparation method conditions

[0065] 1. Selection of the amount of antigen coating on microspheres

[0066] 100 μL of microspheres coded as No. 027 were coated with 1 μg, 3 μg, 6 μg, 9 μg, 12 μg, 15 μg, and 20 μg, respectively. After testing the effect comparison, with 6μg / 1.25×10 6 A microsphere, that is, 12-24ng / 2500-5000 microspheres / test coating, has the best coating effect. After counting under a microscope, store it in a dark place and refrigerate it for later use. Such as figure 1 As shown, the No. 044 microspheres coated with SARS-CoV N protein antigen all fell in the correct detection area, and obtained high signal-to-noise ratio results (MFI value was much greater than 2000), indicating that the optimized suspension chip detection system can be successfully used For the detection of SARS antibodies.

[0067] 2. Optimization of biotinylated antibodies

[0068] The present invention uses amino-active biotin su...

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Abstract

The invention relates to a new method for quantitatively detecting SARS antibody of protein suspended chip in SARS antibody and a product thereof. The method of the invention has good detection ability, high flexibility, strong specificity and wide dynamic range, and establishes open detection modelization platform detected by virus antibody protein suspended chip using SARS antibody as representative.

Description

technical field [0001] The invention relates to a novel quantitative detection method and product for detecting SARS antibodies in serum samples and a preparation method thereof. Background technique [0002] SARS coronavirus (SARS-CoV), the pathogen of severe acute respiratory syndrome (Severe Acute Respiratory Syndrome, SARS, infectious atypical pneumonia) that ravaged the world from the winter of 2002 to the spring of 2003, is a kind of Coronaviridae, a genus of Coronaviridae . Coronavirus is one of the main pathogens of the common cold in adults. The infection rate in children is relatively high, mainly upper respiratory tract infection, and generally rarely affects the lower respiratory tract. In addition, it can also cause acute gastroenteritis in infants and newborns. The main symptoms are watery stools, fever, and vomiting, which can be passed more than 10 times a day. . SARS-CoV is a single-stranded positive-sense RNA virus belonging to the order Nidoviridae, the...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543G01N33/546G01N21/64
Inventor 王静杨宇孙肖红胡孔新张晓龙
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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